226 PHT Lab2 Staining techniques Identification of Bacteria

226 PHT Lab#2 Staining techniques

Identification of Bacteria q Microscopical Examination: • Examination of wet mount preparation. • Examination of stained preparation. q Macroscopical Examination: • Characters of colonies. • Hemolysis on blood agar. • Pigment production. 2

Identification of Bacteria q. Biochemical Tests. q. Additional Tests: • such as serological tests 3

Staining of Bacteria • Bacteria cells are almost colorless, and for this reason a staining technique is often applied to the cells to color them so that their shape and size can be easily determined under the microscope. 4

Staining of Bacteria • Types of staining technique: Simple staining (use of a single stain) For visualization of morphological shape & arrangement. Differential staining (use of two contrasting stain) Identification Gram stain Visualization of structure Acid fast stain Spore stain Capsule stain 5

Staining of Bacteria • Principle of staining: ØDye are generally salts in which one of the ions is colored. ØExample: methylene blue (simple dye) is the salt of methylene blue chloride (MBC) + MBC MB + C ØDyes may be either: ØAcidic dyes [ -ve] ØBasic dyes [ +ve] 6

Indirect staining with acidic dye (Negative staining) • The negative stain technique does not • • • stain the bacteria but stain the background. The bacteria will appear clear against a dark background. No heat fixation or strong chemicals are used, so the bacteria less distorted than in other staining procedure. Example: Nigrosine are acidic stain (negatively charged), so the –ve stain doesn’t stain the bacteria due to ionic repulsion of bacterial cell wall 7

Preparation and Fixation of Bacteria for Staining (Preparation of Smear) • Objective: To kill the microorganism &fix them to the slide to prevent them from being washed out during the process of staining. 8

Preparing a smear for staining. (The following procedure is used for all of our staining) 1. Flame (sterilize) your inoculating loop/needle before and after use. Heat from base to tip. Be sure to get the entire wire red hot. Make sure that you are collecting your hair

. 2. Prepare the smear a. With solid culture (agar colony), place a small drop of distilled water on a clean slide. Drag the sterile inoculating needle tip through the edge of an isolated colony. Gently spread the mixture into a circle the size of a quarter. b. With liquid culture 3. loop Letofthe smear (A liquid cultureair candry be completely. Do not apply directly heat while because this can lyse placed on thedrying slide and spread out. ) the cells 10

Smear preparation S Fixation 11

Simple Staining • Objective: To show the morphological shapes and arrangement of bacterial cells. a) Direct staining with basic dye: ü Materials: ü Cultures of Staphylococci, Bacillus ü Methylene blue stain 12

Simple Staining • Procedure: - MB 1 -2 min 13

Basic Shapes of Bacteria Cocci Bacilli 14

Arrangements Cocci Irregular Clusters Tetrads Staphylococci Micrococci Chains or Pairs Streptococci 15

Bacterial Arrangement - Clusters (group). - Chains. - Pairs (diploids). - No special arrangement. 16

Results Name of staining technique: Name of dye: Shape of cells: Arrangement of cells: Color: Name of m. o: 17

Simple Staining • Name of stain. tech. : • • • Simple Stain Name of dye: Methylene blue Shape of cells: - bacilli Arrangement of cells: Chinese letter Color: - Blue Name of m. o: - Coryebacteria 18

Simple Staining • Name of stain. tech. : simple stain • Name of stain: - Methylene blue • Shape of cells: - cocci • Arrangement of cells: clusters • Color: - Blue • Name of m. o: Staphylococci 19

Simple Staining • Name of stain. tech. : simple stain • Name of stain: - Crystal violet • Shape of cells: - cocci • Arrangement of cells: clusters • Color: - purple • Name of m. o: Staphylococci 20

Negative staining Candida 21

Negative staining Staphylococci 22

Negative staining Bacillus 23

Principle of Differential Stains * Application of the primary stain. * Decolourization. *Application of the counter-stain. 24

Gram Stain • It is the most important differential stain used in bacteriology because it classified bacteria into two major groups: a) Gram positive: Appears violet after Gram’s stain b) Gram negative: Appears red after Gram’s stain 25

Flaming of Loop 26

Smearing out of the sample 27

Smear Fixation 28

Gram Staining “One of the most common mistakes is to decolorize a smear for too long a time period. Even Gram-positive cells can lose the crystal violet-iodine complex during prolonged decolorization.

Gram Stain Gram-positive bacteria • Have a thick peptidoglycan layer surrounds the cell. • The stain gets trapped into this layer and the bacteria turned violet. • Retain the color of the primary stain (crystal violet) after decolorization with alcohol Gram-negative bacteria • have a thin peptidoglycan layer that does not retain crystal violet stain. • Instead, it has a thick lipid layer which dissolved easily upon decoulorization with Acetone-Alcohol. • Therefore, cells will be counterstained with safranin and turned red. 30

Gram’s +ve Bacteria Gram’s -ve Bacteria 31

Gram Stain • Materials: - • Cultures of Staphylococci, Candida, Bacillus, gram –ve bacteria • Crystal violet (primary stain) • Gram’s iodine (mordant) • Acetone-alcohol (decolorizing agent) • Safranin (counter stain) 32

Gram Staining Technique 33

Gram +ve Staphylococci Gram –ve bacteria Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 34

Step 1: Crystal Violet Step 2: Gram’s Iodine Step 3: Decolorization (Aceton-Alcohol) Step 4: Safranin Red 35

Results: Shape: Cocci Arrangement: clusters Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Staphylococci 36

Results: Shape: Oval Arrangement: Single Colour: Violet Gram’s reaction: Gram’s +ve Name of microorganism: Candida 37

Results: Shape: Bacilli Arrangement: Chains Colour: Violet Gram’s reaction: Gram +ve Name of microorganism: Bacillus 38

Results: Shape: Rods Arrangement: Single Colour: red Gram’s reaction: Gram -ve Name of microorganism: Gram –ve bacteria 39

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